Matrix Metalloproteinase-7 Associated with Congestive Heart Failure in Peritoneal Dialysis Patients: A Prospective Cohort Study

Background Matrix metalloproteinase-7 (MMP7) is markedly expressed in patients with chronic kidney disease; its expression in dialysate and role in patients undergoing peritoneal dialysis (PD) have not been well established. Methods Participants undergoing PD from June 1st, 2015, to June 30th, 2020, were involved and were followed up every 3 months for the first year and every 6 months thereafter until death, PD withdrawal, or the end of the study. Data at each follow-up point were collected and analyzed for the association with congestive heart failure (CHF), PD withdrawal, and combined endpoint. Results A total of 283 participants were included in this study. During a median follow-up of 21 months, 20 (7%) participants died, 93 (33%) withdrew from PD, and 105 (37%) developed CHF. A significantly increased level of serum and dialysate MMP7 was observed at baseline. Dialysate MMP7 presented a good linearity with serum MMP7. Baseline serum and dialysate MMP7 levels were associated with CHF in multivariable Cox proportional hazards regression models. After categorization, participants with high baseline MMP7 levels had a higher incidence of CHF (42%), and the hazard ratios (95% confidence intervals) were 1.595 (1.023-2.488). Interestingly, participants with higher serum MMP7 levels were trended to use dialysate with higher glucose concentration. However, the ultrafiltration volumes were not significantly increased. Higher MMP7 levels were also positively associated with PD withdrawal and combined endpoint. Conclusions The expression of MMP7 in serum and dialysate was markedly increased and was tightly associated with the risk of CHF in PD patients. This finding suggests that the measurement of MMP7 may inform strategies for managing CHF at an earlier stage.


Introduction
With technique development and government medical insurance coverage, more patients with end-stage renal disease (ESRD) have received renal replacement therapy in China, during which more than 100,000 patients have received peritoneal dialysis (PD) [1]. Congestive heart failure (CHF), mainly caused by water retention, remains a common clinical challenge in PD patients, especially in those accompanied by anuria and/or ultrafiltration failure (UFF) [2]. The development of CHF in PD patients will increase the burden for both public health and the economy and lead to a higher frequency of readmission as well as poorer clinical outcomes including cardiovascular events and overall mortality [3][4][5][6]. Except for increasing glucose concentration of dialysate and transferring continuous ambulatory PD (CAPD) to intermittent PD (IPD) or to automatic PD (APD) [7][8][9], there are only a few effective methods to improve CHF. What is more, either exposure to higher glucose dialysate or increasing dialysis frequency might not improve CHF adequately, or some patients have to be transferred to hemodialysis (HD). In addition, irreversible damages had occurred before the clinical diagnosis has been established [10][11][12]. Early recognition and management of CHF in the PD population is an elephant in the room.
Matrix metalloproteinase-7 (MMP7), the smallest secreted MMP, plays an important role in many biological activities [13] and has been detected in the serum of patients with disease states [14][15][16]. MMP7 is significantly increased and acts as a noninvasive biomarker of prefibrotic signaling in patients with chronic kidney disease [17,18]. Recently, Yin et al. found that MMP7 was highly expressed both in the serum and in the fluid discharged after dialysis in PD patients and could upregulate water channel protein-aquaporins 1 (AQP1), thereby enhancing water reabsorption of peritoneal mesothelial cell [19]. Thus, highly expressed MMP7 in the peritoneal dialysate might be associated with water transport across the peritoneal membrane and may be related to some clinical complications such as CHF. To confirm this, a prospective cohort study involving 283 participants was conducted in our department.

Participants and Follow-Up.
This was a single-center, prospective, cohort study. From June 1st, 2015 to June 30th, 2020, all participants who received peritoneal dialysis catheterization after diagnosis of chronic renal failure, with baseline clinical data and serum samples, aged 18 years or older and received PD treatment for more than 3 months, were eligible for this study. Participants who were reluctant to participate in the study or had irregular follow-up (less than once a year after dialysis or never after catheter implantation) were excluded. Follow-up was planned every 3 months for the first year and every 6 months thereafter until death, PD withdrawal, or June 30th, 2020. Healthy volunteers were also recruited and served as healthy control. All participants included in this study have provided written informed consent. The research protocol was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University.

Sample Collection and Measurement.
Serum samples and 24 h peritoneal dialysate samples were obtained at baseline every three months during the first-year follow-up and annually thereafter. Blood samples were also collected from healthy volunteers. At the centrifuged condition of 4°C, 3000 r/min for 10 minutes, all supernatant liquids of samples which labeled with study identification numbers were preserved at −80°C. Samples were thawed at room temperature on the day of measurement and analyzed immediately. MMP7 was measured according to standard protocols in the central laboratory at baseline at the first-year follow-up and annually thereafter by ELISA kit (DY907, R&D Systems, Minneapolis, USA). Based on duplicate samples from study patients, the measured variation ranged from 3 to 8%.   Figure 1). An individual with definitive CHF would exhibit symptoms like shortness of breath or edema and receive 3 Mediators of Inflammation medical treatment for CHF. As determined by two trained physicians, a definite diagnosis of CHF in our study requires that a minimum of one major and two minor or two major criteria be present concurrently [22], as shown in Supplementary Table 1. The secondary outcomes were PD withdrawal (including transfer to hemodialysis, renal transplantation, or death) and combined endpoint (including CHF, other cardiovascular diseases, stroke, or death), mainly based on the clinical diagnosis of the physician. Urine volume less than 100 ml in 24 hours was defined as anuria. UFF is defined as failure to achieve at least 400 ml of net UF during a 4 h dwell using 4.25% glucose.

Statistical Analyses.
Dialysate MMP7 values were naturally log (ln) transformed to normalize the distribution. Continuous variables are shown as means and standard deviations (SD), and categorical variables were summarized as frequency and percentages. Baseline characteristics were compared between the two groups using t-test for continuous variables and the chisquare test for categorical variables. The random effects model was used to analyze the overall comparison of dialysate glucose concentration and UF volume over time between groups. The association between serum and dialysate MMP7 and CHF was assessed by using unadjusted and adjusted (age, sex, BMI, baseline LVEF, baseline UF, baseline total KT/V, loss of renal residual function, and mean dialysate glucose concentration and serum ALB of the first 12 months after dialysis) Cox regression analyses. We next categorized participants into two groups according to baseline serum MMP7 levels (MMP7 ≤ 4:80 ng/ ml, MMP7 > 4:80 ng/ml). The Kaplan-Meier and Cox regression analyses were performed on the created variables. Cox proportional hazards models were also used to analyze the association of serum and dialysate MMP7 levels and PD withdrawal or combined endpoint. The unadjusted and adjusted hazard ratios (HR) were calculated with 95% confidence intervals (95% CI). All analyses were performed with Stata version 15 (Stata Statistical Software: Release 15). A two-sided p value < 0.05 was considered as statistically significant.

Association between MMP7 and CHF.
The association between MMP7 and CHF was analyzed at each follow-up time point. We found that only baseline serum (HR 1.089, 95% CI 1.024-1.159, p = 0:006) and dialysate (HR 1.558, 95% CI 1.212-2.002, p = 0:001) MMP7 levels were associated with CHF (Table 2). After adjusted by demographics (age, sex, and BMI), loss of renal residual function, baseline LVEF, UF volume, total KT/V, mean dialysate glucose concentration and serum ALB of the first 12 months after dialysis, baseline serum, and dialysate MMP7 levels were still significantly associated with CHF (Table 2). MMP7 levels at other follow-up points were not associated with CHF (Supplementary Table 2). As shown in Figures 5(a) and 5(b), participants in the group with the high baseline serum MMP7 levels (>4.80 ng/ml) had a higher incidence of CHF (42%), and the risk of CHF greatly increased with the HR of 1.577 (95% CI 1.067-2.331, p = 0:02) by unadjusted analysis and of 1.595 (95% CI 1.023-2.488, p = 0:04) after adjusted analysis, compared to the lower baseline serum MMP7 group (Table 3).

Relationship between MMP7 and Longitudinal Changes
in Clinical Parameters of PD. Since CHF might be tightly associated with water retention caused by UFF and decreased urine output [23], we next analyzed the relationships between MMP7 and longitudinal changes of UF volume as well as dialysate glucose concentration. Interestingly, participants with higher serum MMP7 levels trended to use dialysate with higher glucose concentrations earlier over time (Figure 6(a)), but the UF volume appeared to be similar between groups with different MMP7 levels ( Figure 6(b)).  (Figure 7). Serum MMP7 levels were associated with PD withdrawal (HR 1.090, 95% CI, 1.028-1.550, p = 0:004) and combined endpoint (HR, 1.083, 95% CI, 1.011-1.580, p = 0:02). A similar correlation was found also on the dialysate MMP7 levels. The results were consistent after adjustment for demographics (age and sex), the mean values of dialysate glucose concentration, urine volume, serum creatinine, serum phosphate, hemoglobin, and KT/V during the first 12 months after dialysis (Table 4).

Discussion
In the prospective cohort study, we demonstrated that MMP7 was highly expressed in the serum and dialysate of participants undergoing PD. The level of serum MMP7 first decreased and then gradually increased with the prolonged PD vintage. Interestingly, baseline serum and dialysate MMP7 levels were associated with CHF. Participants with high baseline serum MMP7 levels had an increased risk of developing CHF with an HR of 1.595 (95% CI 1.023-2.488, p = 0:04). Although participants with higher MMP7 levels trended to use higher glucose concentrations of dialysate, UF volume did not improve significantly. These results suggested that MMP7 might play an important role in PD patients. Some studies have shown that the expression of MMP7 was almost undetectable in healthy people [24,25]. However, its expression was significantly induced in serum and urine in both acute and chronic kidney injury models [26]. Fang et al. found that higher urine MMP7 levels were correlated with an increased risk of progressive acute kidney injury [27]. However, other studies confirmed that urine MMP7 levels were positively associated with renal fibrosis scores and negatively correlated with renal function progression [24,28]. Peritoneal dialysate is incessantly stayed in the peritoneum and is exchanged 4 times a day, which is the crucial way for the clearance of solute and fluid in PD patients. In addition, high glucose exposure could promote the expression of MMP7 [29]. Therefore, we speculated that MMP7 might also be significantly expressed in the peritoneal dialysate. We did find the markedly expressed MMP7 in both serum and dialysate of PD patients. The quite welllinear association between MMP7 in serum and in dialysate suggests that dialysate MMP7 might be mainly excreted from serum.
Functionally, MMP7 could degrade a variety of extracellular matrix components, activate proteases, and play a key   Mediators of Inflammation role in regulating diverse cellular processes such as matrix remodeling and epithelial-mesenchymal transformation (EMT) [14,30]. However, the expression and role of MMP7 in the dialysate have not been well established. Our previous cross-sectional study had shown that dialysate MMP7 was negatively correlated with UF volume in PD patients [19]. The reduction of UF volume might be directly related to water retention, leading to the occurrence of CHF. We then focused on the association between MMP7 and CHF. Due to sufficient patient-doctor interactions in our cohort, participants were able to report their symptoms and signs of CHF to the physicians in charge timely and could be admitted to the hospital for early intervention. These might account for the seemingly high number of hospitalizations for CHF. Interestingly, both baseline serum and dialysate MMP7 did associate with CHF even in the fully adjusted model. When categorized into two groups, participants with high MMP7 levels had a higher risk of developing CHF, which was 1.595 times higher than that in the group with low MMP7 levels. These data indicated that the markedly increased baseline MMP7 might be a potential early marker for CHF.   Mediators of Inflammation    Figure 7: Kaplan-Meier curves for PD withdrawal and combined endpoint. Participants were categorized into two groups according to baseline serum MMP7 levels (MMP7cat = 1, MMP7 ≤ 4:80 ng/ml; MMP7cat = 2, MMP7 > 4:80 ng/ml). The Kaplan-Meier survival function curves showed a higher cumulative incidence of PD withdrawal (p = 0:035) and combined endpoint (p = 0:008) in participants with increasing levels of baseline serum MMP7. PD withdrawal was defined as death, transfer to hemodialysis, or renal transplantation. Combined endpoint was defined as death, CHF, cardiovascular diseases, or stroke. MMP7: matrix metalloproteinase-7; PD: peritoneal dialysis; HR: hazard ratio; CI: confidence interval; CHF: congestive heart failure.

Mediators of Inflammation
Here, we did confirm that MMP7 was associated with CHF, but whether MMP7 is a causal factor of CHF is not sure. Interestingly, we found that the difference in UF volume between the MMP7 groups was similar after a longitudinal study over time, although participants in the high MMP7 group trended to use dialysate with a higher glucose concentration. Initially, due to possible differences in concentrations of serum and intraperitoneal MMP7, serum MMP7 might be excreted into the peritoneal cavity through the dialysate. Serum MMP7 decreased first with time due to the clearance of dialysate. However, the long-term continuous exposure of the peritoneum to high glucose dialysate might have contributed to the nonphysiological elevation of MMP7 in the dialysate. Serum MMP7 tended to increase with the duration of dialysis. In our previous studies, we have found that MMP7 might affect UF volume by altering the permeability of peritoneal mesothelial cells via the water channel protein-AQP1 and leading to cell enlargement. High MMP7 levels were associated with reduced UF volume. Due to the clinical presentation, the treatment for participants with decreased UF volume would be modified including the use of dialysate with higher glucose concentrations, since all decisions were made by the physicians. Nevertheless, its effect on improving UF in participants with high MMP7 was limiting, which might account for the high incidence of CHF events.
The etiology of CHF in PD patients is particularly complex. Atherosclerosis, hypertension, fluid overload, and inadequate dialysis prescription or poor adherence might be the key contributing factors [31][32][33]. Recurrence of CHF increases the readmission rate of PD patients, worsens the prognosis, increases the incidence of mortality and cardiovascular events, and aggravates medical and economic burdens [3-6, 33, 34]. In most patients, the diagnosis can only be established when overt manifestations of CHF are present. Irreversible cardiac damages have occurred before diagnosis [35,36]. Therefore, earlier detection is an urgent clinical problem. In our study, baseline MMP7 levels were tightly associated with CHF, indicating that serum and dialysate MMP7 might act as one of the early biomarkers. For PD patients with higher MMP7 levels, we should pay more attention to the follow-up, especially to the balance of intake and outflow, blood pressure and weight change, clinical symptoms, etc. All in all, the noninvasive detection of MMP7 might help physicians make judgments and conduct clinical interventions earlier to prevent CHF.
The following strengths were shown in our study. Firstly, it was a prospective study in which the outcome of each participant was observed during the follow-up period. Secondly, the levels of MMP7 were measured in our central key laboratory with standard procedures which reduced measurement errors. Most importantly, this study has presented several characteristics of MMP7 in PD patients, including the high expression in dialysate, the tight association with the occurrence of CHF, and the potential early biomarker for CHF. However, several limitations should be considered when accounting for the findings. As our results were reflected in a single-center cohort, validation studies from other populations are needed. If MMP7 levels were measured at each CHF event, the results might be more convincing. Due to the small number of deaths during followup, the association between MMP7 and mortality was not explored. The research on the mechanism should be further strengthened.

Conclusion
This study provided a unique perspective on the expression of MMP7 in patients undergoing PD and identified its association with CHF. If further confirmed, measurement of serum and dialysate MMP7 might help clinicians detect CHF events at an earlier stage, conduct proper treatment strategies, and improve the clinical prognosis of PD patients.

Data Availability
The data used and analyzed in our research are available from the corresponding authors upon request. KT/V was calculated by the formulas mentioned before. PD: peritoneal dialysis; MMP7: matrix metalloproteinase-7; HR: hazard ratio; CI: confidence interval; DKD: diabetic kidney disease; ESRD: end-stage renal disease. a Adjusted with demographics (age and sex), the mean values of dialysate glucose concentration, urine volume, serum creatinine, serum phosphate, hemoglobin, and KT/V during the first 12 months after dialysis. b Adjusted with demographics (age and sex), DKD to ESRD, the mean values of dialysate glucose concentration, urine volume, serum albumin, serum phosphate, hemoglobin, and KT/V during the first 12 months after dialysis. 8 Mediators of Inflammation